Profiling Glycosyltransferase Activity on the Epithelial-Mesenchymal Transition in Breast Epithelial Cells using Peptide Asrrays and Mass Spectrometry High-throughput biotechnology has enabled discovery of genetic and proteomic profiles that identify cancer subtypes with distinct phenotypes. However, similar efforts to profil based on the posttranslational modifications mediated by glycosyltransferases have lagged due to lack of efficient high-throughput assays. Such enzymatic profiling technology is needed because these enzymes exert dramatic effects on cell phenotype. For example, fibronectin glycosylation by GalNac-T3 and GalNac-T6 in prostate cells epithelial cells is essential for the TGF-beta induced epithelial-mesenchymal transition (EMT), a developmental process that cancer cells hijack to enable metastasis. This example is particularly relevant to breast cancer because this form of fibronectin, onfFN, is upregulated in breast cancer tissue and not present in normal mammary tissue. Moreover, overexpression of GalNac-T6 induce an EMT-like phenotype in MCF10A, an immortalized breast epithelial cell line. Despite these evidence it remains unclear if GalNAc-T3 and GalNAc-T6 regulate EMT in breast tissue by a similar mechanism as that in prostate cells. Therefore my hypothesis that GalNAc-T3 and GalNAc-T6 promote EMT in breast cells via glycosylation of fibronectin and that their activity can serve as novel biomarkers of EMT. This proposal seeks to investigate the role of GalNAc-T3/-T6 on TGF-beta EMT in breast cells and use an innovative technology to profile their activity directly from cell lysate, known as SAMDI mass spectrometry. This method uses peptide substrate arrays to profile enzyme activities in a sample and has demonstrated effectiveness for studying several enzymatic chemistries, including glycosylation. Preliminary data further demonstrate that SAMDI profiling is feasible via detection endogenous galactosyltransferase activity in the triple-negativ breast cancer cell line, MDA-MB-231. The first aim will entail synthesis of a peptide library that can distinguish activities between GalNAc-T3 and GalNAc-T6 and then test by SAMDI whether GalNAc-T3 and GalNac-T6 activities can be used to profile EMT. In the second aim, I will investigate the extent to which GalNAc-T3 and -T6 regulate EMT in breast epithelial cells. Using overexpression and knockdown studies, I will test whether EMT is dependent on the expression of GalNAc-T3/-T6 and whether EMT is dependent on the presence of onfFN. This proposal aims to address two critical needs: fill a gap in available technology relevant to the field of glycobiology as a whole as well as provide fundamental information important to the breast cancer community.